DESCRIPTION: The long-term goal of this proposal is to increase the understanding of the molecular mechanism underlying Mendelian inherited deficiencies of mitochondrial multienzyme complexes. The model system chosen for study is maple syrup urine disease (MSUD), an inborn error in branched-chain amino acid metabolism that produces mental retardation and often fatal ketoacidosis. The enzyme deficient in MSUD, the mitochondrial branched-chain -keto acid dehydrogenase (BCKD) complex, is organized around a 24-meric transacylase (E2) core, to which other enzyme components comprising a thiamine pyrophosphate (TTP)-dependent decarboxylase (E1), a lipoamide dehydrogenase (E3), a specific kinase and a specific phosphatase are attached through ionic interactions. MSUD is genetically heterogeneous and mutations in four (E1-alpha, E1-beta, E2, and E3) of the six genetic loci have been described. However, the mechanisms by which MSUD mutations perturb protein-protein interactions and macromolecular assembly is poorly understood. These investigators have recently established a correlation between deficiency in the E2 subunit and the thiamine-responsive phenotype in MSUD patients. They have shown that bacterial chaperonins GroEL and GroES, homologs of mitochondrial Hsp60 and Hsp10, respectively, promote folding and assembly of D1 and D2 components of the mammalian BCKD complex. They also report that a subset of MSUD mutations at the E1-alpha locus affect assembly of the E1 component. In this application, they propose to: 1) investigate the TPP-binding fold in the E1 component by characterizing critical residues, including those affected in MSUD, for the cofactor binding, 2) decipher the biochemical mechanisms for the thiamine-responsive phenotype in E1-deficient MSUD patients, 3) elucidate in vitro the chaperonin-mediated folding of E1-alpha and E1-beta subunits that leads the assembly into the E1 heterotetramer and the effect of MSUD mutations on this process and 4) study chaperonin (Hsp60)-facilitated assembly and degradation of mature E1-alpha and E1-beta subunits inside mitochondria. They propose that information obtained from the proposed research should open new avenues for more effective treatment of this severe metabolic disease, and shed light on chaperonin-mediated biogenesis of mitochondrial multienzyme complexes.